Compositions, formulations and methods of treating preeclampsia-type disorders of pregnancy

ABSTRACT

This invention discloses a method of and composition for treating a PE-type disorder in a subject in need of such treatment comprising administering to the subject a pharmaceutical composition containing a therapeutically effective amount of a TTR polypeptide in admixture with a pharmaceutically acceptable vehicle.

BACKGROUND

Globally, preeclampsia (PE) and other hypertensive disorders ofpregnancy are a leading cause of maternal and infant illness and death.By conservative estimates, these disorders are responsible for 76,000maternal and 500,000 infant deaths each year, from 7-8% of allpregnancies (www.preeclampsia.org). Typically, preeclampsia is diagnosedin the late 2nd or 3rd trimesters, after 20 weeks gestation, though itspathogenisis may occur earlier. Preeclampsia, HELLP Syndrome andeclampsia are manifestations of the same syndrome. Id. PE presents withmaternal symptoms of global endothelial disease, includingglomeruloendotheliosis, liver and cerebral vascularitis. It occurs onlyduring pregnancy and the postpartum period and affects both the motherand the unborn baby. It is a rapidly progressive condition characterizedby high blood pressure (>140) and the presence of proteinuria (>0.3μm/ml) and general edema. Swelling, sudden weight gain, headaches andchanges in vision can also be symptomatic. However, some women withrapidly advancing disease report few symptoms. It is believed to be asystemic disorder associated with a cascade of events and symptoms,including impaired trophoblast invasion, decreased placental perfusion,placental ischemia, oxidative stress and imbalance in angiogenic andprothrombotic factors which can lead to apoptosis of trophoblasts.⁽¹⁻⁴⁾Studies have also reported that in preeclampsia, there are elevatedlevels of circulating or placental TNFα, IL-6, IL-8, IFNγ, leptin, aperturbed renin angiotensin system, complement split products,antibodies to phospholipids, sFlt-1, soluble endoglin, IL-12, decreasedIL-10, NO, and hypoxia⁽⁵⁻¹³⁾ amongst a host of other factors.Uteroplacental abnormalities can result in shallow placentation, poorspiral artery remodeling and placental ischemia. PE is believed to be aplacental condition which resolves after pregnancy terminates/delivery.

Efforts have been made to provide assays for the diagnosis of PE.Numerous assays employ identification and/or measurement of variousbiochemical markers such as specific protein or nucleic acids inmaternal samples.¹⁴ Of these types of assays, noteworthy are thosesuggesting the use of transthyretin (hereinafter “TTR,” formerly calledprealbumin)) as a biomarker.¹⁵

In some instances of PE, labor is induced if the fetus has reached agestational age of at least 37 weeks. If the pregnancy is premature,treatment focuses on allowing the fetus to mature as much as possiblebefore inducing labor and avoiding progression of the disease and/orcomplications by close patient monitoring either by hospitalization orin an outpatient setting. The health of the mother is constantly weighedagainst the health of the fetus and labor induced when one or both arein danger of dying. In some cases, the fetus must be deliveredimmediately, regardless of gestational age, to save the mother's and/orfetus' lives.

A pharmaceutical composition for therapeutic intervention in PE andPE-type disorders would be a significant improvement in treatment.

SUMMARY OF THE INVENTION

We have discovered that administration of TTR to mammals exhibitingsymptoms of preeclampsia alleviates the symptoms of this disorder.

The nucleotide sequence of TTR (identified by accession no.NM.sub.--000371) is disclosed in, e.g., Fex et al., 1979, “Interactionbetween prealbumin and retinol-binding protein studied by affinitychromatography, gel filtration and two-phase partition,” published inEur. J. Biochem. 99 (2), 353-360; Mita et al., 1984, “Cloning andsequence analysis of cDNA for human prealbumin,” published in Biochem.Biophys. Res. Commun. 124 (2), 558-564, and the amino acid sequence ofTTR (identified by accession nos. AAH05310, AAP35853) is disclosed in,e.g., Kanda et al., “The amino acid sequence of human plasmaprealbumin,” J. Biol. Chem. 249: 6796-6805, each of which isincorporated by reference herein in its entirety.

Because eclampsia and HELLP syndrome are manifestations of the samesyndrome, administration of TTR should likewise treat and alleviate thesymptoms of these disorders. Hereinafter, these three disorderscollectively are referred to as PE-type disorders.

TTR is a known 55 kDa protein, a homotetramer with a dimer of dimersconfiguration. Each monomer is a 127-residue polypeptide rich in betasheet structure. Association of two monomers forms an extended betasandwich. Further association of another identical set of monomersproduces the homotetrameric structure. The two thyroxine binding sitesper tetramer sit at the interface between the latter set of dimers.Human and other mammalian TTR DNA sequences have been isolated¹⁶⁻¹⁷ butto our knowledge TTR has not been employed as a pharmaceuticalcomposition to treat PE-type disorders, or in a method for treatingPE-type disorders. It has been suggested that increasing cerebral TTRsynthesis is a potential therapeutic/prophylactic approach to humanAlzheimer's disease¹⁸, and the brains of mice have been treated in vitrowith human TTR at a concentration of 3 μM.¹⁹ It also has been suggestedthat formulations containing unspecified concentrations of TTR protein,fragments or mimics can be made and used to treat amyotrophic lateralsclerosis.20 It is believed that the endogenous ligand for this proteinis thyroxine T4.

Small molecules such as diclofenac or aspirin also are believed tostabilize the structure. In some embodiments small molecules includingcertain amino acids like tryptophan analogs or anti-oxidants and thatare pregnancy compatible would improve pregnancy complications andprovide unique new therapeutic opportunity to preeclampsia market. It isbelieved that such small molecules may fit into the ligand binding site.Again, without being bound by any particular theory, it is believed thatthe presence of ligands can stabilize the protein structures and canprevent mis-foldings and aggregation of TTR protein.

In particular embodiments of this invention it is contemplated that theTTR, subunits or fragments thereof, or non-pathogenic TTR mutants(collectively “TTR polypeptide”) are useful used in a pharmaceuticalpreparations or formulations to treat the PE-type disorders. In someinstances subunits or non-pathogenic TTR mutants are established bystabilization of TTR tetrameric native structure, purified tohomogeneity from cell sources or produced recombinantly orsynthetically.

Accordingly, in one aspect, the invention comprises a method of treatinga PE-type disorder in a mammal, preferably human, subject comprisingadministering to the patient a pharmaceutical formulation of acomposition containing a therapeutically effective amount of a TTRpolypeptide. By “therapeutically effective amount” is meant an amount ofTTR polypeptide (alone or in combination with other drugs) that iseffective in rescuing (i.e., preventing or arresting) abnormalendothelial-trophoblast cross-talk that is a hallmark of PE-typedisorders. As to cross-talk, normal pregnancy serum will exhibittube-vacuole formation of over about 40 tubes/well of a 48 well plate(for example between about 45 and 75 tubes/well. This average number ofsuch tubes-vacuoles in response to normal pregnancy serum is defined as“normal endothelial-trophoblast cross-talk”. In contrast, serum orplasma from a pregnant female at risk for or having preeclampsia willexhibit tube-vacuole formation substantially less than about 40 tubesper well (for example between about 5 and 35 tubes/well). This averagenumber of such tubes-vacuoles is defined as “abnormalendothelial-trophoblast crosstalk”. See Example 2 infra, U.S. PatentApplication Ser. No. 61/063491 filed Feb. 4, 2008 and Kalkunte et al.,In Vitro and In Vivo Evidence for Lack of Vascular Remodeling by ThirdTrimester Trophoblasts, Placenta 29: 871-78 (2008) (PCT PatentApplication No. PCT/US2009/000708). The PE-type disorder may bepreeclampsia, Eclampsia or HELLP syndrome. The therapeutically effectiveamount of TTR polypeptide may comprise a TTR tetramer or a TTR subunit,active fragments thereof, or modified version thereof in admixture witha pharmaceutically acceptable vehicle.

In another aspect, the invention comprises PE-type disorder rescuingtherapeutic compositions of TTR polypeptides. Such compositions comprisea therapeutically effective amount of a TTR polypeptide in admixturewith a pharmaceutically acceptable vehicle. Such compositions can besystemically administered parenterally, intravenously or subcutaneously.When systemically administered, the pharmaceutical formulation forsystemic administration is in the form of a pyrogen-free, parenterallyacceptable aqueous solution. The preparation of pharmaceuticallyacceptable protein solutions or formulations, having due regard to pH,isotonicity, stability and the like, is within the skill in the art.

The invention also comprises preparations of a TTR polypeptide suitablefor oral delivery. Suitable oral formulations may be prepared as anaqueous-based oral solution, or may comprise TTR polypeptide in the formof a gel, a suspension, a lozenge, a pill, a capsule or a coated oruncoated tablet.

In another aspect, the TTR polypeptides may be administered incompositions and formulations with one or more non-steroidalanti-inflammatory compositions (“NSAIDs”). Without being bound by anyparticular theory it is believed that NSAIDs exert their effect throughbinding to T4 binding pockets in TTR. Exemplary non-steroidalanti-inflammatory compositions include diclofenac, flufenamic acid,diflunisal and aspirin.

The dosage regimen involved in the method of treating PE-type conditionswill be determined by the attending physician considering variousfactors that modify the action of drugs, for example the conditions,body weight, and diet of the patient, the severity of conditions, timeof administration and other clinical factors. Generally, the dailyregimen should be in the range of 50-500 micrograms of polypeptide perkilogram of body weight. Particular reference is made to dosages of50-100 mg/kg, 25-50 mg/kg, and 20 mg/kg of recombinant or isolated humantransthyretin protein over a 24 hour period. Further reference made todosages of 10 mg/kg per day administered over a 24 hour period incombination with an NSAID such as diclofenac (10:1 mole/mole).

Since the half life of endogenous TTR is approximately 2 days, thetreatment regimen can be titrated by measuring the serum levels of TTRby ELISA, and the dose adjusted accordingly by the attending physician.As the therapeutic method may also include co-administration with othercompounds, in such cases, the dosage recited above would be adjusted tocompensate for such additional components in the therapeuticcomposition.

The instant invention comprises a method of treating a PE-type disorder(e.g. preeclampsia, eclampsia and HELLP syndrome) in a subject in needof such treatment comprising administering to the subject apharmaceutical composition containing a therapeutically effective amountof a TTR polypeptide in admixture with a pharmaceutically acceptablevehicle. In some embodiments of the method TTR polypeptide comprisesTTR,H—PTGTGESKAPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASGKTSE-NH—CH₂—CH₂—S—CH₂—COELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTAND-NH—CH₂—CH₂—S—CH₂—CO—PRRYTIAALLSPYSYSTTAWTNPKE-OH,andCl—Ac-ELHGLTTEEEFVEGIYKVEIDTKSYWK-ALGISPFHEHAEVVFTAND-NH—CH₂—CH₂—S—CH₂—CO—PRRYTIAALLSPYSYSTTAVVTNPKE-OH.In particular embodiments the therapeutically effective amount of TTRpolypeptide is between about 50 and 500 micrograms per kilogram bodyweight and optionally about 50-100 mg/kg, with particular reference to25-50 mg/kg. Further contemplated is administration over a 24 hourperiod as well as co-administration with a non-steroidalanti-inflammatory (NSAID).

In some embodiments TTR polypeptide is co-administered with NSAID at amolar ratio of TTR polypeptide to NSAID of from about 10:1 to about 1:1.Noted is co-administration wherein the non-steroidal anti-inflammatorycomposition is selected from the group consisting of diclofenac,flufenamic acid, diflunisal and aspirin. Specific embodiments of themethod include co-administration of a TTR polypeptide and NSAIDcomprises within a dosage range of from about 5 to about 100 mg/kg/day.Noted in the employment of such method is the additional step ofmonitoring the level of serum transthyretin in said subject subsequentto said administration. Further noted is the method of employing thepresent composition therapeutically in treatment of kidney pathology,glomerular endotheliosis and excess excretion of protein or proteinuricdisorders.

The invention yet further includes a therapeutic composition comprisinga therapeutically effective amount of a TTR polypeptide in apharmaceutically acceptable vehicle, optionally in the form of apyrogen-free, parenterally acceptable aqueous solution for systemicadministration. Noted as to such composition is TTR polypeptide in anamount of about 3.5 to about 500 mg. Additionally noted is TTRpolypeptide is selected from the group consisting of recombinant TTR,mutants of TTR, synthetic TTR, pharmaceutically active fragments of TTR.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Shows a proteomic analysis using SELDI-TOF. Human serum fromnormal pregnancy and preeclamptic pregnancy were subjected to proteomicanalysis using SELDI-TOF. The data as shown depicts that a protein ofmolecular weight of 13,500 is significantly reduced in preeclampsiaserum as compared to normal pregnancy serum. Data analyses usingsuitable protein database suggests that this protein is transthyretin.Horizontal units are treatment groups at the molecular weight (expressedas Daltons) indicated. Vertical units are the relative intensity (RI) ofthe molecular weight peak in a sample. Different dots representsdifferent patients serum samples (as indicated by N). This figure onlyshows a abnormal molecular weight region, although the analysis includeda range from 3000 to 200,000.

FIG. 2 presents histological data on recombinant transthyretindose-dependently rescue of severe PE serum induced disrupted EC-HTR8crosstalk in tube assay. This figure also shows data on severepreeclampsia or sPE (representing samples L1012, L13, L2, L3 L17,L1020).

FIGS. 2A, B and C illustrate that recombinant TTR dose dependentlyrescues severe PE serum induced disrupted EC-HTR8 crosstalk. NPS standsfor normal serum and sPE for severe preeclampsia serum. The data showserum from severe preeclampsia patients (sPE) (Panel B and C,photographs #5 & 9) but not normal pregnancy (NPS) (Panel A, photograph#1) inhibits the interaction between crosstalk of cellular partners'viz., endothelial cells (EC) and trophoblasts (HTR). Recombinant TTR indose dependent manner rescues sPE serum induced disruption (Panels B andC, photographs #6-8 & 10-12). Please note representative photographs ofsPE samples from two different patients are shown in Panel B & C.

FIG. 3 presents data on recombinant transthyretin dose-dependent rescueof mild PE (mPE) serum induced disrupted EC-HTR8 crosstalk in tubeassay. Tested were L1033, L9, L1029, L1022, L19, L5.

FIGS. 3D and E illustrate that recombinant TTR dose dependently rescuesmild PE serum induced disrupted EC-HTR8 crosstalk. NPS stands for normalserum and mPE for mild preeclampsia serum. The data show serum from mildpreeclampsia patients (mPE) (Panel D and E, #13 & 17) inhibits theinteraction between crosstalk cellular partners viz., endothelial cells(EC) and trophoblasts (HTR). Recombinant TTR in dose dependent mannerrescues mPE serum induced disruption (Panels D and E, #14-16 & 18-20).Please note representative photographs of mPE samples from two differentpatients are shown in Panel D & E.

FIG. 4 is a graphic reproduction of the quantification of in vitrocross-talk and rescue results as described in Example 2.

FIG. 5 shows the in vivo confirmation of the rescue of pregnancy byrecombinant TTR. Briefly, a single intra-peritoneal injection of 100 μlof serum from preeclampsia patients (sPE) but not normal pregnancy (NPS)in pregnant IL10 knockout mice induced intra-uterine growth restrictionas shown by the fetal size in picture (A) and in the fetal weight graph(B), hypertension as seen by systolic blood pressure (C) and proteinuria(D). Importantly, co-administration of recombinant TTR rescues sPE seruminduced anomalies (A-D) at different doses.

FIG. 6 shows the SEDI-TOF data of serum samples obtained from mouse fromdifferent treatment groups. As shown here, severe preeclampsia seruminduces reduction of TTR levels in mouse which is then rescued byrecombinant TTR administration as indicated by rising levels of TTRexpressed as relative intensities.

FIG. 7 presents in vivo experimental data to confirm that when TTRactivity is abolished using a neutralizing antibody, normal pregnancyserum

(NPS) behaves like preeclampsia serum in that it causes intrauterinegrowth restriction as represented by the fetal weight graph (A) andcause hypertension as represented by the increase in systolic bloodpressure seen in graph (B). Isotype control antibody (IgY) is used toshow the specificity of the TTR neutralizing antibody. Note that **indicates statistical significance at probability of greater than 95%confidence levels as compared to NPS group (expressed as P<0.05).

FIG. 8 presents in vivo experimental data to confirm that when TTRactivity is abolished using a neutralizing antibody, normal pregnancyserum (NPS) behaves like preeclampsia serum in that it causesproteinuria. Isotype control antibody is used to show the specificity ofthe TTR neutralizing antibody. These findings further confirm thatreduced TTR levels in serum can cause preeclampsia like symptoms and useof recombinant TTR can rescue the symptoms.

FIG. 9 data is based on an in vitro experiment. With neutralized TTRusing a neutralizing antibody, normal pregnancy serum (NPS) behaves likepreeclampsia serum in that it inhibits the interaction of endothelialcells and trophoblasts as seen in NPS+TTR antibody panels. These valueshave been quantified and shown graphically in FIG. 9 a

DETAILED DESCRIPTION EXAMPLE 1 Proteomic Analysis of Normal versus PESerum

The early detection of biomarkers associated with preeclampsia wouldsignificantly decrease morbidity and mortality from this pregnancycomplication but such early detection is difficult in the absence ofphysical symptoms, which tend to present later in pregnancy. Resultsclearly indicate that preeclampsia serum is different from normalpregnancy serum in its pathology-inducing properties. A massspectrometry SELDI-TOF proteomics approach was used to determine whichprotein molecules are dysregulated (i.e., by loss of quantity and/orfunction or by overwhelming presence).

In order to detect low concentration proteins in a sample replete withhighly abundant proteins, a separation of the low and high concentrationproteins is useful. To accomplish this, different fractionationtechniques based on diverse loading platforms can be employed. Forexample, protein binding to hydrophobic beads and their gradual elutionmay be employed. Alternatively, anion exchangers after pHvalue-dependent elution can be used.²¹ Serum from normal and PE humanpatients was used for initial optimization of the experimental settings.The serum was analyzed in native form and analyzed after fractionationin one single procedure using 4 different coated protein chips: ananionic exchanging surface chip (Q10), a cationic exchanging surfacechip (CM10), a hydrophobic surface chip (HSO) and a copper (II) ioncoated surface chip (IMAC30). For data generation, the IMAC30 chip wasthe one which allowed detection of the highest number of differentiallyexpressed proteins.

First, the protein chips were activated in accordance with themanufacturer's protocol. The IMAC30 protein chips were loaded with 50 μlof IMAC charging 100 mM CuSO₄ solution and incubated for 10 minutesfollowed by washing with deionized water with 2 minutes incubation,repeated twice. This was followed by incubation with IMAC-neutralizingbuffer (100 mM sodium acetate pH 4.0) repeated twice, then incubationwith IMAC-binding buffer (100 mM sodium phosphate, 0.5 M sodiumchloride, pH 7.0) for 5 minutes. Thereafter, the normal and PE serumsamples were applied to the chips and incubated for 30 minutes in ahumid chamber at room temperature. Room temperature will be understoodto mean about 20° C. to about 25° C. Each application spot was thenwashed three times with binding buffer to avoid non-specific binding,followed by 2 washings with deionized water to remove salts. After airdrying, 1 μl of sinapinic acid (SPA) dissolved in 50% acetonitrile and0.05% trifluoroacetic acid (TFA) was applied twice to every spot. Thespots were dried between the applications.

The cationic exchange protein chip CM10 was first rehydrated byincubation with CM-low-stringency binding buffer (100 mM sodium acetate,pH 4.0) or CM-high-stringency binding buffer (50 mM HEPES, pH 7.0) for 5minutes and this rehydration step was then repeated once. Thereafter,the normal and PE serum samples were applied to the chips and incubatedfor 30 minutes in a humid chamber at room temperature. Afterwards, thespots were washed 3 times with binding buffer for 5 minutes and twicewith deionized water. After air drying, SPA was applied on the spots asdescribed above.

SELDI-TOF measurements were then performed on all of the chips using theProteinChip System, Series 4000 SELDI-TOF mass spectrometer (CiphergenBiosystems). Calibration was performed externally using the protein MWstandard kit (Ciphergen Biosystems). Ionization of the proteins wasaffected with a laser shot energy of 2200 nJ in the case of proteins <20kDa (low mass range) and 3500 nJ for proteins >20 kDa (high mass range).The final spectra of a spot was generated by combining the spectra of320 laser shots. The bioinformatical analysis was performed usingCiphergen Express Client 3.0 software.

The result of this analysis indicated that number of preeclampsia serumsamples (n=53) had a deficiency in a protein having an approximatemolecular weight of 14 kDa (FIG. 1).

EXAMPLE 2 In Vitro Assay for TTR

The in vitro assay described in this example is based on the premisethat during normal pregnancy, fetal derived trophoblasts called the“invasive cytotrophoblasts” invade the maternal decidua and theendothelial cell lined spiral arteries. This phenomenon can berestructured using serum from pregnancy as physiological milieu andculturing human umbilical vein endothelial cells and trophoblasts onbasement membrane such as matrigel. In response to serum from normalpregnancy, endothelial cells and trophoblasts form a network of tubes asshown in FIG. 2 (Panel A #1). However, serum from pregnancycomplications such as preeclampsia disrupts this cross-talk suggesting“disruptive factors” in serum resulting in poor invasion oftrophoblasts. For additional information see U.S. Provisional PatentApplication No. 61/063491 filed Feb. 4, 2008 and/or Kalkunte, In Vitroand In Vivo Evidence for Lack of Vascular Remodeling by Third TrimesterTrophoblasts, Placenta 29: 871-78 (2008).

Recombinantly produced TTR (detected by SDS-PAGE electrophoresis andpurity 96%) was obtained from AbD Serotec, Raleigh, N.C. 27604, USA.Recombinant TTR obtained in lyophilized form was reconstituted withsterile phosphate-saline buffer to obtain a final concentration of 1mg/ml and maintained at >pH 7.0. The solutions were stored as aliquotsfrozen at −80° C. until use. Thirty minutes prior to experimentation,the aliquots of TTR were thawed, then mixed with serum and incubated at37° C. for 30 minutes.

A. Rescue of Severe PE Serum

The in vitro method further includes incubating a co-culture of humanendothelial cells and human trophoblast cells in the presence of serumor plasma obtained from a pregnant female for a period of timesufficient to permit vacuolization (also referred to as “capillaryformation” and “tube formation” in FIGS. 2-4), and after incubationdetermining whether substantial vacuolization in the co-culture hasoccurred by quantification as described in Example 3.

Blood samples were obtained from normal pregnancy human subjects andpreeclamptic human subjects during first (6-12 weeks), second (13-20weeks) or third (21-40 weeks) trimester of pregnancy and serum separatedroutinely. Pregnancies were considered normal when there were no medicalcomplications.

Preeclampsia was defined when blood pressure was >140/90 mm Hg at leaston two occasion 4 hours to 1 week apart and with proteinuria >300milligram in 24 hr urine collection. Preeclampsia can be classified asmild or severe. Severe preeclampsia is characterized by (1) a systolicblood pressure greater than 160 mm Hg or diastolic blood pressuregreater than 110 mm Hg on 2 occasions at least 6 hours apart in a womanon bed rest and (2) the presence of significant proteinuria. Markedproteinuria is defined as 5 g or more of protein in a 24-hour urinecollection. Severe preeclampsia, at times, may be associated witholigouria, cerebral or visual disturbances, pulmonary edema or cyanosis,epigastric or right upper quadrant abdominal pain, impaired liverfunction, and thrombocytopenia. In mild preeclampsia (or moderate PE),hypertension and proteinuria are present, but not to these extremelevels, and the patient has no evidence of other organ dysfunction. (seehttp://www.emedicine.com/med/topic1905.htm Preeclampsia (Toxemia ofPregnancy). Exclusion criteria were chronic hypertension, diabetes,antiphospholipid antibody syndrome, thrombophillic anomalies, antepartumand postpartum complications.

During pregnancy, angiogenesis is characterized by spiral arteryremodeling for which trophoblast invasion into these maternal bloodvessels is a prerequisite. The data of FIG. 2 show an in vitro model ofinteraction of trophoblasts and endothelial cells that mimics spiralartery remodeling. Specifically, 2.5×104 endothelial cells labeled redand 2.5×10⁴ trophoblasts, labeled green, were co-cultured on matrigelcoated plates and stimulated with either NPS (normal pregnancy serum) orsevere PES (Preeclampsia Serum) as described above. Exemplary resultsare shown in FIG. 2. In the absence of exogenous TTR, serum from severe(FIG. 2 panel B #5 and C #9) preeclampsia patients blocks the“cross-talk” between endothelial-trophoblast cells, causing obviousdifferences in architecture as compared to the same cells stimulatedunder the same conditions with NPS serum (sample L1045; FIG. 2 panel A,#1). Preincubation by mixing of recombinant TTR (AbD Serotec,purity >96%) at different concentrations (0.1, 1.0, 10 microgram/ml ofserum) substantially rescues the “abnormal endothelial-trophoblastcross-talk” and is shown in FIG. 2 panels B & C (#6-8 & 10-12). Therewas no significant effect on NPS mediated tube formation (FIG. 2, PanelA, #2-4). These cells in these panels exhibit more normal architectureand increases in capillary tube formation.

The number of vacuoles (or tubes) formed per sample were counted.Exemplary results are shown in the graphic panel in FIG. 4. As comparedto the control NPS, the cells co-cultured with either mild or severe PEserum exhibited significant decreases in capillary tube formation (60versus 18 and 14). Statistical significance of experimental differenceswas assessed using Student's paired t-test. The differences wereconsidered to be statistically significant when the p value was <0.05.As shown in FIG. 4, adding TTR dose dependently increase the number ofcapillary tubes formed, resulting in the rescue of the abnormalendothelial-trophoblast cross-talk.

B. Rescue of Mild PE Serum

In mild preeclampsia (or moderate PE), hypertension and proteinuria arepresent, but not to these extreme levels, and the patient has noevidence of other organ dysfunction.

Briefly, 2.5×10⁴ endothelial cells labeled red and 2.5×10⁴ trophoblasts,each from different trimesters labeled green (FIG. 2), were co-culturedon matrigel coated plates and stimulated with either NPS or mild PES asdescribed above.

In the absence of exogenous TTR, serum from mild (sample L1033 and L9;FIG. 3 panel D photograph #13, and Panel E, photograph #17) preeclampsiapatients blocks the “cross-talk” between endothelial-trophoblast cells,causing obvious differences in architecture as compared to the samecells stimulated under the same conditions with NPS serum (sample L1045;FIG. 2 panel A, photograph #1). Pre-incubation by mixing of recombinantTTR (AbD Serotec, purity >96%) at different concentrations (0.1, 1.0, 10μg/ml of serum) substantially rescues the “abnormalendothelial-trophoblast cross-talk” and is shown in FIG. 3 panels D,photograph #14-16 & Panel E, photograph #18-20. The number of vacuoles(or tubes) formed per sample were counted and represented in the graph(FIG. 4, mPE with and without TTR). As compared to the control NPS, thecells co-cultured with mild PE serum exhibited significant decreases incapillary tubes formation (60 versus 20 and 15). Statisticalsignificance of experimental differences was assessed using Student'spaired t-test. The differences were considered to be statisticallysignificant when the p value was <0.05.

EXAMPLE 3 Quantification of Results

The in vitro studies conducted in Example 2 were quantified by countingthe number of tube like structures termed as “vacuole” underfluorescence microscope (Nikon Eclipse TS 100 coupled with CCD camera)in four different fields at 4× magnification. Each vacuole in thiscontext is the small cavity completely bound by elongated cellularstructure as indicated in FIG. 2 Panel A and FIG. 2 Panel C (shown bybold arrow). As observed under a microscope, vacuolization comprisesthin walled vacuoles having few branch points. The branch points are thepoints from which multiple vacuoles are initiated/connected. Thequantity of tubes formed (also means number of vacuoles) will besubstantially less by comparison with normal pregnancy serum. Normalpregnancy serum will exhibit tube-vacuole formation over about 50-60tubes/well of a 48 well plate. These average numbers of suchtubes-vacuoles in response to normal pregnancy serum are defined as“normal endothelial-trophoblast cross-talk”. In contrast, serum orplasma from a pregnant female at risk for or having preeclampsia willexhibit tube-vacuole formation substantially less under about 50-60tubes per well. These average numbers of such tubes-vacuoles are definedas “abnormal endothelial-trophoblast cross-talk” (FIG. 4). Dosedependent rescue of “abnormal endothelial-trophoblast cross-talk” by TTRadministration is shown in FIG. 4.

EXAMPLE 4 In Vivo Administration of TTR Rescues Preeclampsia likeSymptoms in a Mouse Model

The anti-inflammatory cytokine IL-10 μlays a critical role in pregnancybecause of its regulatory relationship with other intrauterinemodulators and its wide range of immunosuppressive activities.Significantly, its local production by gestational tissues is welldocumented. We have demonstrated that IL-10 expression by the humanplacenta was gestational age-dependent, with significant expressionthrough the second trimester followed by attenuation at term. IL-10expression was also found to be poor in decidual and placental tissuesfrom unexplained spontaneous abortion cases, and from deliveriesassociated with preterm labor and preeclampsia. However, themechanism(s) by which IL-10 protects the fetus remains poorlyunderstood; IL-10^(−/−) (knockout) mice suffer no pregnancy defectsunless challenged with inflammatory agents. Recently we showed thatIL-10^(−/−) mice were more sensitive as compared with wild typecounterparts, to “disruptive factors” in PES and exhibit full spectrumof preeclampsia-like symptoms in response to human PES. See U.S.Provisional Patent Application No. 61/063491 filed Feb. 4, 2008 (PCTPatent Application PCT/US2009/000708). Furthermore, since PE serum isable to disrupt trophoblast and endothelial cell functions, IL-10^(−/−)mice can provide a model system to study the pathogenesis ofpreeclampsia-like symptoms.

Recombinantly produced TTR (detected by SDS-PAGE electrophoresis andpurity 96%) was obtained from AbD Serotec, Raleigh, N.C. 27604, USA.Recombinant TTR obtained in lyophilized form was reconstituted withsterile phosphate-saline buffer to obtain a final concentration of 1mg/ml and maintained at >pH 7.0. The solutions were stored as aliquotsand frozen at −80° C. until use. Thirty minutes prior toexperimentation, the aliquots of TTR were thawed, then mixed with 100 μlof PE serum samples. Pregnant IL-10^(−/−) mice (C57BL/6, Jackson Labs,USA) were then injected intraperitoneally on gestational day (g.d.) 10at a TTR dose of 20 microgram/ mouse. The gestational period in mice is20 days. Similarly, 100 μl PE serum or normal pregnancy serum wasadministered to different set of animals as control. On g.d.16/17, urineand serum samples were collected from each mouse, and blood pressuremeasurements were taken. Blood pressure was taken by an establishedtail-cuff method which utilizes a programmed sphygmomanometer. Theanimals adapted for 5 min using a warming test chamber (IITC LifeScience Inc, Woodland Hills, Calif.) at controlled temperature (35° C.).The measurements were carried out on day 17 of pregnancy using DigiMedblood pressure analyzer, (MicroMed, Louisville, Ky., 40222-4683). Eachmeasurement of blood pressure is an average of three readings at 1 minintervals from a number of animals (˜3-5 each). Systolic blood pressurewas compared among non-pregnant and pregnant mice. All animals were agematched. Data was analyzed using Digi-Med® System Integrator™ Model 400(DMSI-400).

Total urinary albumen was measures using Albumin (mouse) ELISA kit(ALPCO Diagnostics, Salem, N.H.) and urinary creatinine was measuredusing Metra Creatinine Kit (Quidel Corporation, San Diego, Calif.).Protinuria as represented as a ration of urinary albumin to creatinine(expressed as μg/mg). (Baseline values seen in mice range from 100-400μg/mg.)

On g.d.17 the mice were euthanized, the uterine horns were extracted,photographed and pregnancy outcomes were recorded. The results of thestudy as summarized in FIG. 5 suggest that a number of PE serum samplesrepresenting mild and severe phenotypes induced some or allPE-associated symptoms when injected i.p. on g.d. 10 in IL^(−/−) mice.The effects in response to only one administration of serum (100 μl)were evaluated on g.d. 17. Signature PE symptoms including intrauterinegrowth restriction (IUGR) as reflected by reduced fetal weight(representative photographs FIG. 5A and average fetal weights of anumber of fetus(n), FIG. 5B), elevated systolic blood pressure (FIG. 5C)and proteinuria (FIG. 5D) were observed in response to PE serum (sPE)when compared to normal pregnancy serum (NPS). Importantly,co-administration of TTR with sPE significantly reversed these PE likesymptoms in mice (sPE+TTR group in FIG. A-D).

CLINICAL EXAMPLE 1 Severe Preeclampsia

A pregnant woman at week 24 of gestation presents herself for routinecheckup. Blood pressure measurements show systolic blood pressureis >150 mmHg (hypertension) and urine analysis show excess excretion ofprotein and creatinine (proteinuria, >1.5). The patient is diagnosed assevere preeclampsia. The subject is treated with a composition ofrecombinant or isolated human transthyretin protein at a dose of 100mg/kg body weight over a 24 hour period and the levels of serumtransthyretin are monitored by suitable detection method by ELISA or bySELDI-TOF. It is noted that in particular embodiments a dosage of 50-100mg/kg of recombinant or isolated human transthyretin protein aretherapeutic.

CLINICAL EXAMPLE 2 Mild Preeclampsia

A pregnant woman at week 28 of gestation presents herself for routinecheckup. Blood pressure measurements show systolic blood pressureis >140 mmHg (hypertension) and urine analysis by ELISA show highprotein to creatinine ratio (proteinuria) is >0.3, the patient isdiagnosed as mild preeclampsia. The subject is treated with acomposition of transthyretin protein at a dose of 40 mg/kg body weightover a 24 hour period and the levels of serum transthyretin aremonitored by suitable detection method by ELISA or by SELDI-TOF. Theclinical outcome of hypertension and proteinuria are monitored duringthe following week. Depending on the clinical diagnosis, a second doseof 40 mg/kg is given over a 24 hr period. It is noted that in particularembodiments a dosage of 25-50 mg/kg of recombinant or isolated humantransthyretin protein are therapeutic.

CLINICAL EXAMPLE 3 Severe Preeclampsia

A pregnant woman at week 22 of gestation presents herself for routinecheckup. Blood pressure measurements show systolic blood pressureis >155 mmHg (hypertension) and urine analysis show excess excretion ofprotein and creatinine (proteinuria, >1.5), the patient is diagnosed assevere preeclampsia. The subject is treated with a composition oftransthyretin protein in combination with diclofenac 10:1 (mole/mole) ata dose of 10 mg/kg per day administered over a 24 hour period and thelevels of serum transthyretin is monitored by suitable detection methodby ELISA or by SELDI-TOF. The clinical outcome of hypertension andproteinuria are monitored in the following week. Depending on theoutcomes, a second dose of 5-10 mg/kg of transthyretin-diclofenac isadministered over a 24 hr period.

It is noted that in particular embodiments the mole ratio oftransthyretin protein to diclofenac is from about 1:1 to about 10:1. Itis further noted that an initial dosage of about 5-100 mg/kg/day iscontemplated (total transthyretin protein plus NSAID). Without beingbound by any particular theory it is believed that NSAIDs such asdiclofenac act to stabilize transthyretin protein in vivo. Note isfurther made of NSAIDs such as Ibuprofen, Naproxen, Fenoprofen,Ketoprofen, Flurbiprofen, Oxaprozin, Indomethacin, Sulindac, Etodolac,Piroxicam, Meloxicam, Tenoxicam, Droxicam, Lornoxicam, Isoxicam,Mefenamic acid, Meclofenamic acid, Flufenamic acid, Tolfenamic acid,Celecoxib, Rofecoxib Valdecoxib, Parecoxib Lumiracoxib and Etoricoxib

CLINICAL EXAMPLE 4 Severe Preeclampsia-Functional Peptides/Fragments

A pregnant woman at week 26 of gestation presents herself for routinecheckup. Blood pressure measurements show systolic blood pressureis >155 mmHg (hypertension) and urine analysis show excess excretion ofprotein and creatinine (proteinuria, >1.5). The patient is diagnosed assevere preeclampsia. The subject is treated with a compositionconsisting of functional peptides/fragments of synthetic transthyretinprotein at a dose of 50-100 mg; preferably 20 mg/kg per day over a 24hour period, Subsequent to dosing, the patient's levels of serumtransthyretin are monitored by suitable detection method by ELISA or bySELDI-TOF. The clinical outcome of hypertension and proteinuria aremonitored in the following week. Depending on the outcomes, a seconddose of 10-20 mg/kg of said transthyretin composition is given over a 24hour period. Included in said composition are synthetic transthyretin asexemplified byH—PTGTGESKAPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASGKTSE-NH—CH₂—CH₂—S—CH₂—COELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTAND-NH—CH₂—CH₂—S—CH₂—CO—PRRYTIAALLSPYSYSTTAWTNPKE-OHor their fragment peptides exemplified byCl—Ac-ELHGLTTEEEFVEGIYKVEIDTKSYWK—ALGISPFHEHAEVVFTAND-NH—CH₂—CH₂—S—CH₂—CO—PRRYTIAALLSPYSYSTTAVVTNPKE-OHor other non-limiting peptide fragments indicated in the reference“Synthesis of an analog of the thyroid hormone-binding proteintransthyretin via regioselective chemical ligation.” Wilce J A, Love SG, Richardson S J, Alewood P F, Craik D J. J Biol Chem, 2001 Jul. 13;276(28):25997-6003). The composition indicated is also contemplated toinclude biologically active agent fusions of transthyretin such asPEG-TTR (PEG-TTR variant) (incorporated in its entirety US patentapplication 20090191624 Use of transthyretin peptide/protein fussions toincrease the serum half-life of pharmacologically activepeptides/proteins).

CLINICAL EXAMPLE 5 Severe Preeclampsia/HELLP

A pregnant woman at week 24 of gestation presents herself for routinecheckup. Blood pressure measurements show systolic blood pressure of 170mmHg (hypertension) and urine analysis show excess excretion of proteinand creatinine (proteinuria, 7.0), the patient is initially diagnosed assevere preeclampsia. Additionally, the blood analysis for liver functiontest show elevated levels of liver enzymes AST and ALT and elevation inplatelet count. The patient now is confirmed with a diagnosis of HELLPsyndrome. The subject is treated with a composition consisting oftransthyretin protein in combination with aspirin 10:1 mole ratio atdose of 50-100 mg, preferably at 20 mg/kg per day over a 24 hour periodand the levels of serum transthyretin and platelet count is monitored bysuitable detection method. The clinical outcome of hypertension andproteinuria are monitored in the following week. Depending on theoutcomes, a second dose of 10-20 mg/kg of said transthyretin compositionis given over a 24 hr period.

CLINICAL EXAMPLE 6 Severe Preeclampsia

A pregnant woman at week 27 of gestation presents herself for routinecheckup. Blood pressure measurements show systolic blood pressure of 160mmHg (hypertension) and urine analysis show excess excretion of proteinand creatinine (proteinuria, 5.0), the patient is initially diagnosed asexperiencing severe preeclampsia. Additionally, the blood analysis forliver function test show elevated levels of liver enzymes AST and ALTand elevation in platelet count. The patient now has a confirmeddiagnosis of HELLP syndrome. The subject is treated with a compositionconsisting of transthyretin protein stabilized with aspirin (5:1 moleratio) at a dose of 50-100 mg, preferably 20 mg/kg per day over a 24hour period and the levels of serum transthyretin and platelet count aremonitored by suitable detection method. The clinical outcome ofhypertension and proteinuria are monitored in the following week.Depending on the outcomes, a second dose of 10-20 mg/kg of saidtransthyretin composition is given over a 24 hr period.

Contemplated therapeutic compositions for this example are further tocomprise hepatoprotectant (e.g., silymarin, flavobion, thioctacid) andanti-oxidants such as vitamin C, lipoic acid and minerals and vitaminsincluding but not limited to Vitamin B12, Vitamin B3.

CLINICAL EXAMPLE 7 Severe Preeclampsia

A pregnant woman at week 24 of gestation presents herself for routinecheckup. Blood pressure measurements show systolic blood pressureis >160 mmHg (hypertension) and urine analysis show excess excretion ofprotein and creatinine (proteinuria, >4.5), the patient is diagnosed assevere preeclampsia. The patient is treated with a composition oftransthyretin mutant proteins exemplified by mutation in serine residueSer¹¹⁷→Cys (S117C)], glutamine acid residue Glu⁹²→Cys (E92C) orthreonine residue [(T119→Methionine (T119M)] with or without furtherstabilizations at a dose of 50-100 mg/kg body weight over a 24 hourperiod and the levels of serum transthyretin are monitored by suitabledetection method by ELISA or by SELDI-TOF. The serum samples aftertreatment are also monitored for their ability to support theendovascular dual cells tube formation and compared with the disruptiveactivity of the serum samples before treatment. A methodology of usingan in vitro approach is presented in detail in the PCT patentapplication WO/2009/099603.

CLINICAL EXAMPLE 8 Eclampsia

A pregnant woman at week 28 of gestation presents has progressed frompreeclampsia to eclampsia exhibiting tonic-clonic seizures. Bloodpressure measurements show systolic blood pressure is >150 mmHg(hypertension) and urine analysis show excess excretion of protein andcreatinine (proteinuria, >1.5). The patient is diagnosed as eclampsia.The subject is treated with a composition of recombinant or isolatedhuman transthyretin protein at a dose of 100 mg/kg body weight over a 24hour period and the levels of serum transthyretin are monitored bysuitable detection method by ELISA or by SELDI-TOF.

The pharmacologically active compositions of this invention can beprocessed in accordance with conventional methods of Galenic pharmacy toproduce medicinal agents for administration to subjects, e.g., mammalsincluding humans.

The compositions of this invention individually or in combination areemployed in admixture with conventional excipients, i.e.,pharmaceutically acceptable organic or inorganic carrier substancessuitable for parenteral, enteral (e.g., oral or inhalation) or topicalapplication which do not deleteriously react with the activecompositions. Suitable pharmaceutically acceptable carriers include butare not limited to water, salt solutions, alcohols, gum arabic,vegetable oils, benzyl alcohols, polyethylene glycols, gelatin,carbohydrates such as lactose, amylose or starch, magnesium stearate,talc, titanium dioxide, silicic acid, viscous paraffin, perfume oil,fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc.The pharmaceutical preparations can be sterilized and if desired mixedwith auxiliary agents, e.g., lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, flavoring and/or aromatic substances and the likewhich do not deleteriously react with the active compositions. They canalso be combined where desired with other active agents, e.g., vitamins.

In some embodiments of the present invention, dosage forms includeinstructions for the use of such compositions.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants, including suppositories. Ampules,vials, and injector cartridges are convenient unit dosages.

Also for parenteral application, particularly suitable are tablets,dragees, liquids, drops, suppositories, or capsules. A syrup, elixir, orthe like can be used wherein a sweetened vehicle is employed. Sublingualand buccal forms are also noted.

Sustained or directed release compositions can be formulated, e.g.,liposomes or those wherein the active component is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc. It is also possible to freeze-dry the newcompositions and use the lyophilizates obtained, for example, for thepreparation of products for injection.

Generally, the compositions of this invention are dispensed in unitdosage form comprising 3.5-500100 mg of TTR or active fragments or TTRmutations. And in particular embodiments this is combined with NSAIDs ina ration of about 10:1 to about 1:1 (molar) in a pharmaceuticallyacceptable carrier per unit dosage.

All patents, patent applications, publications, and other referencescited herein are hereby incorporated by reference in their entirety.Although the invention has been particularly described with reference tocertain preferred embodiments, artisans of ordinary skill willappreciate that changes in form and details may be made withoutdeparting from the scope of the appended claims.

REFERENCES

-   1. DiFederico et al., Preeclampsia is associated with widespread    apoptosis of placental Cytotrophoblasts within the uterine wall, Am    J Pathol 155:293-301 (1999).-   2. Genebacev et al., Invasive cytotrophoblast apoptosis in    preeclampsia. Hum Reprod 14 (suppl 2):59-66 (1999).-   3. Leung et al., Increased placental apoptosis in pregnancies    complicated by preeclampsia, Am J Obstet Gynecol 184:1249-50 (2001).-   4. Balkundi et al., Labor-associated changes in Fas ligand    expression and function in human placenta. Pediatr Res 47:301-08    (2000).-   5. Venkatesha et al., S9oluble endoglin contributes to the    pathogenesis of preeclampsia. Nat Med 12(6):642-49 (2006).-   6. Peracoli et al., Tumor necrosis factor-alpha in gestation and    puerperium of women with gestational hypertension and preeclampsia,    Am J Reprod Immunol 57(3):177-85 (2007).-   7. Jonsson et al., Cytokine mapping of sera from women with    preeclampsia and normal Opregnancies, J Reprod Immunol 70(1-2):83-91    (2006).-   8. Banerjee et al., Placental expression of interferon-gamma (IFN-γ)    and its receptor IFN-γ R2 fail to switch from early hypoxic to late    normotensive development in preeclampsia, J Clin Endocrinol Metab    90(2):944-52 (2005).-   9. Hendler et al., The levels of leptin, adiponectin, and resistein    in normal weight, overweight, and obese pregnant women with and    without preeclampsia, Am J Obstet Gynecol 193(3 Pt2):979-83 (2005).-   10. Shah, D. M., Role of the renin-angiotensin system in the    pathogenesis of preeclampsia, Am J Physiol Renal Physiol    288(4):F614-25 (2005).-   11. Salmon et al., Antiphospholipid antibodies and pregnancy loss: A    disorder of inflammation, J Reprod Immunol 2008, vol. 77, no 1, pp.    51-56-   12. Girardi et al., Heparin prevents antiphospholipid    antibody-induced fetal loss by inhibiting complement activation, Nat    Med 10(11):1222-26 (2004).-   13. Sharma et al., Leptin, IL-10 and inflammatory markers (TNF-γ,    IL-6 and IL-8) in pre-eclamptic, normotensive pregnant and healthy    non-pregnant women, Am J Reprod Immunol 58(1):21-30 (2007).-   14. U.S. Pat. Nos. 6,735,529; 6,620,590; 6,495,330 and 6,258,540 and    United States Patent Publication Nos. 2007/0185200; 2004/0038305;    2007/0178530; 2007/0104707; 2007/0020766; 2006/0183175 and    2005/0074746.-   15. PCT Patent Publication WO2008/046160 (PCT Application No.    AU2007/001598 filed 19 Oct. 2007 and entitled Assay for the    Detection of Biomarkers Associated with Pregnancy Related    Conditions); Vascotto et al., Oxidized transthyretin in amniotic    fluid as an early marker of preeclampsia, J Proteome Res: 2007, 6    (1), pp 160-170; and Atkinson, K. R. L., Proteomic Biomarker    Discovery for Preeclampsia, Doctor of Philosophy in Biological    Sciences thesis, The University of Auckland, 2008 (which discloses    that upregulation of transthyretin may indicate its utility as a    preeclampsia biomarker). (Page 147)-   16. U.S. Pat. No. 4,816,388 filed 30 Oct. 1985 entitled Human    Prealbumin and Related Methods and Products.-   17. Duan et al., Isolation, characterization, cDNA cloning and gene    expression of an avian transthyretin, Eur J Biochem    200:679-87 (1991) and various other publications cited in the latter    (references 3, 4, and 20-28).-   18. Buxbaum et al., Transthyretin protects Alzheimer's mice form the    behavioral and biochemical effects of Aβ toxicity, Proc Natl Acad    Sci 105:2681-86 (2008).-   19. Stein et al., Neutralization of transthyretin reverses the    neuroprotective effects of secreted amyloid precursor protein (APP)    in APP_(Sw) mice resulting in tau phosphorylation and loss of    hippocampal neurons: support for the amyloid hyopothesis, J Neurosci    24(35):7707-17 (2004).-   20. PCT Patent Publication WO 2006/071469 filed 2 Dec. 2004 entitled    Modulation of the Neuroendocrine System as a Therapy for Motor    Neuron Disease.-   21. Myers et al., Use of proteomic patterns as a novel screening    tool in pre-eclampsia, J Obstet Gynaecol. 24(8):873-4 (2008). Park    et al., Identification of proteomic biomarkers of preeclampsia in    amniotic fluid using SELDI-TOF mass spectrometry. Reprod Sci.    15(5):457-68 (2008).

1. A method of treating a PE-type disorder in a subject in need of such treatment comprising administering to the subject a pharmaceutical composition containing a therapeutically effective amount of a TTR polypeptide in admixture with a pharmaceutically acceptable vehicle.
 2. The method according to claim 1 wherein the preeclampsia-type disorder is preeclampsia.
 3. The method according to claim 1 wherein the preeclampsia-type disorder is eclampsia.
 4. The method according to claim 1 wherein the preeclampsia-type disorder is HELLP syndrome.
 6. The method according to claim 1 wherein said TTR polypeptide comprises TTR.
 7. The method according to claim 1 wherein said TTR polypeptide comprises H—PTGTGESKAPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASGKT SE-NH—CH₂—CH₂—S—CH₂—COELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTAND-NH—CH₂—CH₂—S—CH₂—CO—PRRYTIAALLSPYSYSTTAVVTNPKE-OH.
 8. The method according to claim 1 wherein said TTR peptide comprises fragments that are stable and include synthetic or enzyme breakdown products that mimic or modulate TTR activity or stabilizes native TTR protein.
 9. The method according to claim 1 wherein said therapeutically effective amount of TTR polypeptide is between about 50 and 500 micrograms per kilogram body weight.
 10. The method of claim 1 wherein said therapeutically effective amount of TTR polypeptide is about 50-100 mg/kg.
 11. The method of claim 10 wherein therapeutically effective amount of TTR polypeptide is about 25-50 mg/kg.
 12. The method of claim 1 wherein said therapeutically effective amount of TTR polypeptide is administered over a 24 hour period.
 13. The method according to claim 1 wherein said TTR polypeptide is co-administered with a non-steroidal anti-inflammatory (NSAID).
 14. The method according to claim 13 wherein said TTR polypeptide is co-administered with said NSAID at a molar ratio of TTR polypeptide to NSAID of from about 10:1 to about 1:1.
 15. The method according to claim 1 wherein said TTR polypeptide is a stabilized TTR complex.
 16. The method according to claim 15 wherein said TTR-complex includes stable TTR complexes with NSAIDS, metal cations, small molecular weight compounds including aromatic, heterocyclic, phenolics, arylheterocyclic and their derivatives, amino acids.
 17. The method according to claim 13 wherein the non-steroidal anti-inflammatory composition is selected from the group consisting of diclofenac, flufenamic acid, diflunisal and aspirin.
 18. The method of claim 13 wherein said co-administered amount of a TTR polypeptide and NSAID comprises from about 5 to about 100 mg/kg/day.
 19. The method of claim 1 further comprising the step of monitoring the level of serum transthyretin in said subject subsequent to said administration.
 20. The method according to claim 1 wherein the disorder is a hypertensive disorder.
 21. The method according to claim 1 wherein the disorder is a kidney pathology, glomerular endotheliosis and excess excretion of protein or proteinuric disorders.
 22. The method according to claim 1 further comprising reducing production of soluble anti-angiogenic factor.
 23. The method according to claim 1 wherein the disorder is of placental origin.
 24. The method according to claim 1 wherein said treatment induces pro-angiogenesis.
 25. The method according to claim 1 wherein said need of said subject intrauterine growth restriction.
 26. A therapeutic composition comprising a therapeutically effective amount of a TTR polypeptide in a pharmaceutically acceptable vehicle.
 27. The composition according to claim 26 in the form of a pyrogen-free, parenterally acceptable solution for systemic administration.
 28. The composition according to claim 26 wherein the therapeutically effective amount of a TTR polypeptide is about 3.5 to about 500 mg.
 29. The said composition according to claim 26 wherein said TTR polypeptide is selected from the group consisting of recombinant TTR, mutants of TTR, synthetic TTR, pharmaceutically active fragments of TTR and TTR-complexes. 